Biochemical and Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis Serogroup B

Abstract

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBPdependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin⅐sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.

Protection of Salmonella by ampicillin-resistant Escherichia coli in the presence of otherwise lethal drug concentrations

“…At the same time, co-culturing with S. enterica serovar Typhimurium was associated with increased b-lactamase levels and/or leakiness of the ampicillin-resistant E. coli cells in the presence of ampicillin. It is known that a variety of growing Gram-negative bacteria release membrane blebs (Katsui et al 1982;Kadurugamuwa & Beveridge 1997;Li et al 1998;Beveridge 1999;Post et al 2005). Such blebs are associated with abnormal in-growth of the outer membrane, and they typically appear around the septa during cell division.…”

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

“…This increase in stimulatory potency was not caused by an altered expression of Bvg Ϫ -regulated virulence factors and was shown to be dependent on the enzymatic activity of PagL. It is well established that purified LPS, on a molar basis, is biologically more active than LPS incorporated into membrane structures such as in bacterial cells, outer membrane vesicles, or liposomes (36,42). This can probably be explained by an increased accessibility of LPS in soluble aggregates for extraction and subsequent transfer by LBP/CD14 to the TLR4/MD-2 complex.…”

“…However, by disturbing LPS-LPS interactions, e.g., by EDTA treatment (29), LPS can be released from the outer membrane into the external environment. In previous studies, it was shown that membrane-bound LPS has a lower proinflammatory cytokineinducing activity than soluble LPS aggregates (36,42), probably due to the better accessibility of LPS in soluble aggregates for extraction and subsequent transfer by LBP/CD14 to the TLR4/MD-2 complex. Deacylation of LPS could potentially weaken the LPS-LPS interactions, leading to increased release from the cells and counterbalancing the effect of the reduced endotoxic activity of the purified, deacylated LPS.…”

MD-2–Dependent Pulmonary Immune Responses to Inhaled Lipooligosaccharides

“…These proteins engage endotoxin in a series of sequential protein-endotoxin and protein-protein interactions (8). LBP catalyzes the transfer of LPS from bacterial membranes (10) or aggregates of purified LPS to CD-14 either in soluble form or membrane-bound via a glycosyl-phosphatidylinositol (GPI)-linked membrane anchor (11). Monomeric endotoxin:CD-14 complexes that are released are the preferred substrate form of endotoxin for MD-2, presented either in extracellular form (6,7) or membrane bound via association with TLR4 (9).…”